Abstract

Dietary estrogens, such as lignans, are similar in structure to
endogenous sex steroid hormones and may act in vivo to
alter hormone metabolism and subsequent cancer risk. The objective of
this study was to examine the effect of dietary intake of a lignan-rich
plant food (flaxseed) on urinary lignan excretion in postmenopausal
women. This randomized, cross-over trial consisted of three 7-week
feeding periods during which 31 healthy postmenopausal women, ages
52–82 years, consumed their habitual diets plus 0, 5, or 10 grams of
ground flaxseed per day. Urine samples collected for 2 consecutive days
during the last week of each feeding period were analyzed for lignan
content (enterodiol, enterolactone, and matairesinol) by isotope
dilution gas chromatography/mass spectrometry. Compared with the 0-gram
flaxseed diet, consumption of 5 or 10 grams of flaxseed significantly
increased excretion of enterodiol by 1,009 and 2,867 nmol/day,
respectively; significantly increased excretion of enterolactone by
21,242 and 52,826 nmol/day, respectively; and significantly increased
excretion of total lignans (enterodiol + enterolactone + matairesinol)
by 24,333 and 60,640 nmol/day, respectively. Excretion of matairesinol
was not significantly altered by flaxseed consumption.
Consumption of flax, a significant source of dietary estrogens, in
addition to their habitual diets increased excretion of enterodiol and
enterolactone, but not matairesinol, in a dose-dependent manner in this
group of postmenopausal women. Urinary excretion of lignan metabolites
is a dose-dependent biomarker of flaxseed intake within the context of
a habitual diet.

Introduction

Dietary estrogens, or phytoestrogens, are compounds found in
plants and plant products that possess some estrogenic or
antiestrogenic activity (1, 2, 3)
, and growing evidence
suggests that these phytoestrogens may play a role in cancer prevention
(2, 4, 5, 6, 7)
. Lignans, one type of phytoestrogen, are
diphenolic compounds similar in structure to endogenous sex steroid
hormones (Fig. 1)⇓
and are hypothesized to act in vivo to alter hormone
metabolism and subsequent cancer risk (1, 4, 5, 6, 7, 8)
. Flaxseed
is the most concentrated food source of the plant lignan
secoisolariciresinol, which is converted by the colonic microflora to
the mammalian lignan enterodiol (9, 10, 11, 12)
. Enterodiol can
then be oxidized by the colonic microflora to form the mammalian lignan
enterolactone (11, 13, 14)
. Flaxseed also contains small
quantities of the plant lignan matairesinol, which is also converted by
the colonic microflora to enterolactone (11, 13, 14)
.

Flaxseed has been shown to reduce the early risk markers for and
incidence of mammary and colonic carcinogenesis in animal models
(15, 16, 17, 18)
and to affect menstrual cycle length in
premenopausal women (8)
. Enterolactone and enterodiol, the
mammalian lignans excreted in response to flaxseed consumption
(19, 20, 21, 22)
, have also been shown to reduce early markers for
and incidence of mammary carcinogenesis in animal models (23, 24), decrease cell proliferation
(25, 26, 27)
, increase concentrations of sex hormone-binding
globulin (1, 28, 29, 30)
and inhibit the activity of three
enzymes [aromatase (31, 32)
, 5α-reductase
(33)
, and 17β-hydroxysteroid dehydrogenase
(33)]
that play key roles in sex hormone metabolism.

Although there have been numerous studies examining the effects of
various diets or dietary components on lignan excretion in a variety of
populations (1, 34, 35, 36, 37, 38, 39, 40)
and the effects of flaxseed
consumption on lignan excretion in men and premenopausal women
(19, 20, 21, 22)
, to our knowledge, there have been no studies
examining the effects of flaxseed consumption on urinary lignan
excretion in postmenopausal women. The objective of this study was to
examine the effect of dietary intake of a lignan-rich plant food
(flaxseed) on urinary lignan excretion in postmenopausal women
consuming a semicontrolled diet.

Materials and Methods

Subjects.

Healthy postmenopausal women were recruited from the Monastery of the
Sisters of the Order of St. Benedict in central Minnesota. Potential
subjects were screened with a detailed dietary and medical
questionnaire designed to exclude those who had gastrointestinal
disorders, food allergies, or alcohol intake of >2 drinks/day
(equivalent to 720 ml of beer, 240 ml of wine, or 90 ml of hard
liquor); smoked; had taken antibiotics within the last 6 months; had
used hormone replacement therapy; or had dietary habits that were not
representative of the general population (e.g., exclusion of
an entire food group from their diet, <20% or >45% of energy from
fat, or consumption of more than three servings of flax or products
containing flaxseed per week). After screening, 34 subjects were
contacted and agreed to participate by providing informed written
consent. The Institutional Review Board Human Subjects Committee at the
University of Minnesota approved the study. All subjects were healthy
nonsmokers and were at least 1 year postmenopausal as determined by
subjects’ report of the date of their last menses occurring at least 1
year previous to the start of the study. Seven subjects were not taking
any prescription medications; however, due to the age group of the
subjects, it was not possible to exclude for all medications. Of
the remaining 27 subjects, 10 were taking thyroid replacement
medications, 9 were taking antihypertensive medications, 7 were taking
antihyperlipidemic medications, 6 were taking diuretics, 3 were taking
antidepressants, and 1 was taking a corticosteroid. In all cases,
medication use was consistent throughout the duration of the study. All
of the subjects in this study were nulliparous.

Of the 34 subjects who began the study, 32 completed all feeding
periods. One subject withdrew from the study when she moved to another
state, and another withdrew for medical reasons unrelated to study
participation. During the study, one subject underwent antibiotic
therapy, and her results are not included in the statistical analyses
reported here. Only the results from the 31 subjects who completed the
entire study were included in statistical analyses. The mean values
(±SD) for age, height, weight, and
BMI4
of the subjects were 66.9 ± 8.2 years, 162.2 ± 5.2 cm,
64.6 ± 12.2 kg, and 24.7 ± 4.1 kg/m2,
respectively.

Experimental Design.

This study was designed as a randomized, cross-over trial consisting of
three 7-week feeding periods with a 7-week washout period between the
first and second feeding periods and a 14-week washout between the
second and third feeding period. One of the three feeding periods was
used as a control period during which the subjects consumed only their
usual diets. During the remaining two feeding periods, subjects
consumed their usual diets plus either 5 or 10 grams of ground flaxseed
each day. The 5-gram flaxseed supplement provided approximately 25
kcal, 1.2 grams of protein, 1.8 grams of fat (50–60% α-linolenic
acid), 1.4 grams of carbohydrate, and 1.1 grams of dietary fiber,
including 0.6 gram of soluble fiber. The 10-gram flaxseed supplement
provided double these amounts. During the flaxseed-supplemented feeding
periods, the subjects were supplied daily with one tube containing 5 or
10 grams of ground flaxseed. The flaxseed was kept frozen at −20°C
until consumption, and subjects were instructed to consume the contents
of one tube daily in raw form. The subjects typically consumed the
flaxseed in one serving at breakfast. Used tubes were collected, and
any uneaten flaxseed was measured to monitor subject compliance.
Subjects were asked to maintain their usual body weight and diet and
exercise habits throughout the study.

The ground flaxseed was prepared weekly from commercially available
whole flaxseed (Frontier Whole Flax Seed, Norway, IA). The whole
flaxseed was ground to a coarse texture in a household blender for 1
min. The ground flaxseed was then aliquoted immediately into 5- or
10-gram doses and frozen at −20°C. To determine the approximate
plant lignan content of the ground flaxseed, the
secoisolariciresinol-diglycoside content was measured by
high-performance liquid chromatography (41)
by Kenneth
D. R. Setchell (Children’s Hospital Medical Center, Cincinnati, OH).
According to the analysis, the 5- and 10-gram doses of ground flaxseed
provided 10 and 20 mg of secoisolariciresinol (2 mg or 5.5μ
m secoisolariciresinol/gram ground flaxseed),
respectively. The other primary plant lignan, matairesinol, was not
measured because it comprises only about 0.3% of the total lignans in
flaxseed (42)
.

Sample Collection and Analysis.

During the last week of each feeding period, subjects completed
self-reported 3-day diet records to monitor food intake, body weights
were measured, and 24 h urine samples were collected on 2
consecutive days of the 3-day diet record period. Each urine sample was
collected separately in individual plastic tubs containing 200–350 mg
of ascorbic acid and stored at 4°C until processed. The amount of
ascorbic acid per tub was estimated to provide a final concentration of
approximately 1 gram/liter when all urine collections from a 24-h
period were combined. However, due to variability in urine volume and
frequency, actual ascorbic acid concentrations averaged 1.3
grams/liter. The final volume of each 24 h urine collection was
recorded, and an aliquot from each 24 h urine sample was frozen
for subsequent creatinine analysis to monitor the completeness of the
collection. Pooled aliquots of the two 24 h urine collections were
then stored at −20°C after the addition of 10% sodium azide (final
w/v, 0.1% sodium azide).

The urine samples were analyzed for lignan (enterodiol, enterolactone,
and matairesinol) content by isotope dilution gas chromatography-mass
spectrometry according to the method developed by Adlercreutz et
al.(43)
. The urine was first extracted using Bond
Elute LRC C-18 columns (Chrom Tech, Apple Valley, MN) and
further purified on a DEAE-Sephadex A-25 (Sigma Chemical Co., St.
Louis, MO) anion-exchange column in the acetate form. Deuterated
internal standards of the compounds [provided by Dr. T. Hase, Dr. K.
Wähälä, and T. Mäkelä (Department of
Chemistry, University of Helsinki, Helsinki, Finland) in collaboration
with Dr. H. Adlercreutz (Department of Clinical Chemistry, University
of Helsinki, Helsinki, Finland) and Dr. J. Lampe, Cancer Prevention
Research Program, Fred Hutchinson Cancer Research Center, Seattle,
WA)] were added. The conjugates were hydrolyzed, isolated using
Bond Elute C-18 columns, and applied to a QAE-Sephadex A-25 (Sigma
Chemical Co.) anion-exchange column in the acetate form. Two fractions
were collected; the first fraction contained the lignans (enterodiol,
enterolactone, and matairesinol) that were purified further on a
QAE-Sephadex A-25 anion-exchange column in the carbonate form.
Trimethyl-silyl derivatives of the samples were analyzed by gas
chromatography-mass spectrometry in the selective ion-monitoring mode.
The second fraction contained isoflavones, and those results are not
reported in this study.

The urine samples were analyzed singly, in batches, with all of the
samples from one subject run in the same batch. Two quality control
urine samples were included with each batch. The mean values and mean
intra-assay imprecision for the quality control urine samples were as
follows: (a) enterodiol, 3,108 nmol/day (CV, 2.7%);
(b) enterolactone, 43,904 nmol/day (CV, 10.4%); and
(c) matairesinol, 48 nmol/day (CV, 12.6%).

Diet analyses were performed using the Minnesota Nutrition Data System
software (Food Database version 6A, Nutrient Database 21, 1992)
developed by the Nutrition Coordinating Center, University of Minnesota
(Minneapolis, MN; Ref. 44
).

Statistical Analysis.

Statistical analyses were performed using the Statistical Analysis
System (SAS Proprietary Software Release 6.11; SAS Institute, Cary,
NC). Results were analyzed using a repeated-measure ANOVA within
subject. To correct for data that were not normally distributed,
urinary lignan excretion analyses and P value computations
were performed on a logarithmic scale. The linear regression analyses
of enterodiol, enterolactone, and total lignans were performed on the
logarithmic scale, adjusted for subject, and regressed on the amount of
flaxseed in the diet. For reporting purposes, data summaries were
transformed back to the original scale. For all measurements, results
were considered statistically significant at P < 0.05.

Results

Body weight measurements and BMI at the end of each feeding period
are presented in Table 1⇓
. Because the subjects maintained their body weights throughout the
duration of the study, there were no significant differences in these
measurements between any of the feeding periods.

Intakes of total energy, carbohydrate, protein, fat, and dietary fiber
during each feeding period are presented in Table 2⇓
. There were no significant differences in total energy, carbohydrate,
protein, fat, or total fiber intake between any of the feeding periods.
For soluble fiber, intakes for the 5-gram flaxseed feeding period
(9.0 ± 2.2 grams) and the 10-gram flaxseed feeding period
(9.3 ± 2.3 grams) were significantly higher than that for control
(8.3 ± 2.0 grams; P = 0.0358 and
P = 0.0028, respectively).

The ratio of enterodiol:enterolactone excretion did not change with
flaxseed consumption. The mean enterodiol:enterolactone excretion ratio
was 0.16 ± 0.15 (mean ± SD) on the subjects’ habitual
diet, 0.19 ± 0.41 on the 5-gram flaxseed diet, and 0.17 ±
0.31 on the 10-gram flaxseed diet. Of the 31 women, 19 showed a
decrease and 6 showed an increase in the ratio of
enterodiol:enterolactone excretion with flaxseed feeding. Of the
remaining six women, two had an increase in the
enterodiol:enterolactone excretion ratio on the 5-gram flaxseed diet
but a decrease on the 10-gram flaxseed diet, three had a decrease in
the ratio on the 5-gram flaxseed diet and an increase on the 10-gram
flaxseed diet, and the remaining subject had no change in the ratio on
the 5-gram flaxseed diet and an increase on the 10-gram flaxseed diet.

Discussion

Consumption of flaxseed, a significant source of plant lignans, in
addition to their habitual diets significantly increased the excretion
of enterodiol, enterolactone, and total lignans, but not matairesinol,
in a linear, dose-response manner in this group of postmenopausal
women. The results of this study suggest that urinary excretion of
lignan metabolites can be used as a dose-dependent biomarker of
flaxseed intake, and potentially other plant food intake, within the
context of a semicontrolled diet.

Mean urinary enterolactone excretion by subjects in this study while
consuming their habitual diets was comparable to that reported for
postmenopausal omnivorous women (3284 nmol/day; Ref. 34
)
and postmenopausal vegetarian women (3180 nmol/day) living in the
Boston area (45)
, premenopausal omnivores (3160 nmol/day)
in Minnesota (20)
, and premenopausal lacto-vegetarians
living in Helsinki (3650 nmol/day; Ref. 35
). Mean urinary
enterodiol excretion by subjects in this study was also similar to that
of the postmenopausal vegetarian women (400 nmol/day) in Boston and
premenopausal lacto-vegetarians (368 nmol/day) in Helsinki but was
lower than that of the premenopausal omnivores (1090 nmol/day) in
Minnesota. Excretion of enterolactone and enterodiol in this group of
postmenopausal omnivores may be comparable to that of the
postmenopausal vegetarians and premenopausal lacto-vegetarians due to
the similarities in fiber intake. The plant lignan precursors of
enterolactone and enterodiol are believed to be constituents of the
fiber component of plants typically found in the roots and rhizomes and
in the woody parts, stems, leaves, seeds, and fruits (40)
.
In grains, they are more likely localized in the bran layer and in the
aleuronic layer of the grain right below the bran (4, 46)
.
In several studies, Adlercreutz et al.(1, 4, 29, 34)
have reported positive correlations between total fiber
intake and enterolactone, enterodiol, and total mammalian lignan
excretion in a variety of populations. The subjects in this study
consumed an average of 23 grams of dietary fiber each day, similar to
the consumption reported for the postmenopausal vegetarians (24
grams/day) and higher than that of the postmenopausal omnivores (15
grams/day) in the Boston area. However, despite similar enterolactone
excretion between the postmenopausal omnivores in this study and the
premenopausal omnivores in Minnesota, the premenopausal omnivores had
one-third of the dietary fiber intake (8 grams/day) of the subjects in
this study. Therefore, factors other than dietary fiber intake may be
related to the formation and excretion of enterolactone and enterodiol
in humans.

Wide ranges in the excretion of total mammalian lignans have been
observed on various habitual diets in both animals and humans (1, 34, 35, 36, 37, 38, 39, 40)
and in relation to a flaxseed challenge
(19, 20, 21, 22)
. We observed the same variations in excretion in
this group of postmenopausal women (Table 3)⇓
who were consuming similar
but not identical diets. Several researchers have proposed that
differences in the concentrations of lignans excreted may be due to the
composition of the colonic microflora (11, 40, 47, 48, 49)
,
differences in intestinal transit time (11, 47)
, or the
redox level of the large intestine (11)
, all factors
related to the habitual diets of the subjects (1, 36, 40, 49)
. Factors such as these may account for the variations in
excretion observed in our study, despite the fact that these subjects
have stable, long-standing habitual dietary patterns. The subjects in
this study consume foods prepared in a central kitchen and served in a
central dining room and do not frequently leave the monastery to eat
elsewhere, and food items are offered based on a 7-week cycle menu that
has been used for many years. Although the specific foods chosen at
each meal vary among the subjects, the variation is much less than that
encountered in the general population. Therefore, we hypothesized that
the variation in mammalian lignan excretion would be less in this group
of subjects than that which has been reported for other populations
(1, 34, 35, 36, 37, 38, 39, 40)
. However, mammalian lignan excretion
did vary greatly between the subjects, even on the subjects’ habitual
diets, and the variation increased with the addition of flaxseed (Table 3)⇓
.

Researchers have also proposed that the variability observed in lignan
excretion in humans may be due to the existence of several alternate
pathways for lignan metabolism (40, 49, 50, 51)
or the
existence of mammalian lignan metabolites that are not routinely
measured (52)
. In a recent report, Jacobs et
al.(52)
identified six enterolactone metabolites and
three enterodiol metabolites in the urine of two male and two female
subjects. Although they did not quantify the excretion of these
metabolites, the existence of these metabolites supports the hypothesis
that additional lignan metabolites that are not routinely measured do
exist. Additional studies that address the effects of these
metabolites in vivo and the effects of gastrointestinal
function and colonic microfloral differences on lignan metabolism are
needed.

We observed a significant dose-response increase in enterolactone,
enterodiol, and total lignan excretion in this group of postmenopausal
women (Table 3)⇓
. Nesbitt et al.(21)
also
reported a dose-dependent increase in urinary lignan excretion
(P ≤ 0.001, r2 = 0.5184) in
response to the consumption of 5, 15, or 25 grams of raw flaxseed in a
group of premenopausal women. However, Nesbitt et al.(21)
reported that enterodiol was the mammalian lignan
produced in the highest concentration after flaxseed consumption. The
results reported by Nesbitt et al.(21)
are in
agreement with a study by Cunnane et al.(19)
that reported that enterodiol was excreted in greater concentrations
than enterolactone with the consumption of 50 grams of flaxseed/day. In
contrast, enterolactone was excreted in much higher concentrations than
enterodiol in our study and in flaxseed feeding studies by Shultz
et al.(22)
and Lampe et al.(20)
, although the primary plant lignan in flaxseed is
secoisolariciresinol, the direct precursor of enterodiol. Research
studies have suggested that enterodiol is synthesized from
secoisolariciresinol (9, 10, 11, 12)
by facultative anaerobes in
the colon and can be further oxidized by the colonic microflora to
enterolactone (11, 13, 14)
. The conflicting results
reported by these studies may be due to differences in the metabolism
of plant lignans by individuals, a hypothesis supported by the results
of Kirkman et al.(37)
, who reported gender
differences in the excretion of enterolactone and enterodiol on
controlled diets supplemented with vegetables or soy. However, the
studies by Cunnane et al.(19)
and Lampe
et al.(53)
reported that lignan excretion did
not differ significantly between men and women.

Physiological implications of the wide variation in enterodiol and
enterolactone excretion and the differences in the
enterodiol:enterolactone ratio are not currently known. However,
researchers have found differences in the biological activity of these
mammalian lignans. Enterolactone has approximately 10 times the
estrogenic activity of enterodiol (2)
, was more effective
than enterodiol at inhibiting the growth of MCF-7 breast cancer cells
(54)
and LS174T, Caco-2, HCT-15, and T-84 human
colon tumor cell lines (27)
, and more effectively
displaced both estradiol and testosterone binding by sex
hormone-binding globulin (30)
. Enterolactone was also more
effective than enterodiol at inhibiting human placental aromatase
(31, 32)
, 5α-reductase (33)
, and
17β-hydroxysteroid dehydrogenase activity (33)
. These
results suggest that enterolactone may be more effective against the
prevention of some types of cancer, in particular, colon cancer and
hormone-dependent breast cancer, than enterodiol. Therefore, subjects
who excrete higher concentrations of enterolactone and presumably have
higher plasma enterolactone concentrations may have more protection
against hormone-dependent cancers than subjects who excrete higher
concentrations of enterodiol.

In conclusion, consumption of 5 or 10 grams of flaxseed by
postmenopausal women on a semicontrolled diet increased the excretion
of enterolactone, enterodiol, and total lignans in a
dose-dependent manner without changing the ratio of
enterodiol:enterolactone excretion, suggesting that urinary excretion
of lignan metabolites can be used as a dose-dependent biomarker of
flaxseed consumption. Because flaxseed and its mammalian lignan
products enterolactone and, to a lesser extent, enterodiol have been
shown to influence the early risk markers for and incidence of mammary
and colonic carcinogenesis in animal models, decrease cell
proliferation in vitro, and influence factors that effect
the hormone concentrations in humans, increases in the metabolism and
excretion of these compounds may offer increased protection against
hormone-dependent cancers. Wide variations in the excretion of
enterodiol, enterolactone, and total lignans and the
enterodiol:enterolactone ratio suggest that the factors that may
influence individual variations in mammalian lignan formation,
absorption, and excretion need to be examined so that we may have a
better understanding of the metabolism of these compounds.

Acknowledgments

We thank the Sisters of the Order of St. Benedict (St. Joseph,
MN) for their participation in, dedication to, and support of this
research study; Dr. Kenneth D. R. Setchell for conducting the lignan
analysis of the ground flaxseed; Carol Haggans for grinding and
weighing the flaxseed, Desireé Nichols and Michelle Ethun for
entering dietary intake data, and Michelle Ethun, Laura Marti, and
Bridgette Wagener for distributing flax and collecting diet records and
urine samples.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by NIH National Cancer Institute Grant
R01CA-66675-01 and by the 1998 Jean Hankin Nutritional Epidemiology
Award from the American Dietetic Association Foundation.